Journal: bioRxiv

Article Title: Conversion rate to the secondary conformation state in the binding mode of SARS-CoV-2 spike protein to human ACE2 may predict infectivity efficacy of the underlying virus mutant

doi: 10.1101/2021.07.14.452313

Figure Lengend Snippet: SPR-experiment testing for the presence of a two state reaction. 500 nM SARS-CoV-2 S1-S2 spike protein was injected over a constant immobilization level of 25 RU hACE2 and normalized to 100 % RU for the time point of injection phase end. Injection times were gradually increased (150 – 600 s).

Article Snippet: The biotinylated hACE2-fc (residues 18-740) was purchased by Acrobiosystems and contains a C-terminal IgG1 Fc-Tag (residues 100-330), followed by a biotinylated Avitag.

Techniques: Injection

Journal: bioRxiv

Article Title: Conversion rate to the secondary conformation state in the binding mode of SARS-CoV-2 spike protein to human ACE2 may predict infectivity efficacy of the underlying virus mutant

doi: 10.1101/2021.07.14.452313

Figure Lengend Snippet: (A) Multicycle experiment with SARS-CoV-2 RBD. hACE2-fc was immobilized on a protein A/G sensor chip and SARS-CoV-2 RBD was injected in concentration range of 3.9 – 1000 nM. The K D was globally fitted with a 1:1 Langmuir based interaction model. The kinetic parameters were determined with K D of 21.3 nM, k a of 4.25 E+5 +/- 2.2 E+2 [1/Ms] and k d of 9.1 E-3 +/- 4.2E-6 [1/s]. ( B ) Test on secondary state reaction. hACE2-fc was immobilized on a protein A sensor surface and 500 nM of SARS-CoV-2 was injected at a constant concentration for increasing contact intervals. Time points of injection phase end were normalized to 100 % and the dissociation starting point was aligned on the time scale.

Article Snippet: The biotinylated hACE2-fc (residues 18-740) was purchased by Acrobiosystems and contains a C-terminal IgG1 Fc-Tag (residues 100-330), followed by a biotinylated Avitag.

Techniques: Injection, Concentration Assay

Journal: bioRxiv

Article Title: Conversion rate to the secondary conformation state in the binding mode of SARS-CoV-2 spike protein to human ACE2 may predict infectivity efficacy of the underlying virus mutant

doi: 10.1101/2021.07.14.452313

Figure Lengend Snippet: (A) hACE2-fc was immobilized on a protein A/G sensor surface and CoV trimer proteins were injected in concentration range of 0.62 – 50 nM. The sensograms were globally fitted with the secondary state reaction model. (SARS-CoV 2002) : K D1 6.7 nM, k a1 6.72 E+5 ± 4.4E+3 [1/Ms], k d1 4.68 E-3 [1/s] ± 6.9E-5, k a2 8.80 E-3 ± 6.9E-5 [1/Ms], k d2 2.07E-4 ± 1.60 E-6 [1/s]; K D-total 160 pM (SARS-CoV-2 wt) : K D1 1.8 nM, k a1 7.82 E+5 ± 1.2E+3 [1/Ms], k d1 1.41 E-3 ± 3.75E-5 [1/s], k a2 1.05E-2 ± 1.8E-4 [1/Ms], k d2 2.44E-4 ± 3.5E-6 [1/s]; K D-total 41 pM. ( SARS-CoV-2 B.1.1.7 ) K D1 192.7 nM, k a1 1.31 E+6 ± 1.5E+4 [1/Ms], k d1 2.5 E-2 ± 1.5E-3 [1/s], k a2 5.3E-2 ± 1.5E-3 [1/Ms], k d2 1.1 E-4 ± 2.6E-6 [1/s]; K D-total 40 pM. For graphic representation of the distribution of primary and secondary state reaction, a component analysis was performed for each of the trimeric spike proteins using the injection concentration of 5.56 nM spike. The sensogram (total) is composed of the primary binding event (red), followed by a secondary transition event (blue) which results in a highly stable secondary complex ( B ) On–off chart for the kinetic values of the SARS-CoV spike trimers in the primary and secondary state.

Article Snippet: The biotinylated hACE2-fc (residues 18-740) was purchased by Acrobiosystems and contains a C-terminal IgG1 Fc-Tag (residues 100-330), followed by a biotinylated Avitag.

Techniques: Injection, Concentration Assay, Binding Assay

Journal: bioRxiv

Article Title: SARS-CoV-2 spike D614G variant confers enhanced replication and transmissibility

doi: 10.1101/2020.10.27.357558

Figure Lengend Snippet: (a) Affinity between S1 and hACE2 determined by Bio-layer interferometry. Biotinylated S1 protein (S1–614D or S1–614G) was loaded onto surface of streptavidin biosensors. Association was conducted using hACE2 protein followed by dissociation. (b) Binding of Fc-tagged or polyhistidine-tagged S1 to BHK-hACE2 cells is shown as peaks of fluorescence detected by flow cytometry. (c) Replication kinetics of recombinant viruses in (left) Vero E6 at 37°C and (right) hNE at 33°C. Supernatant was collected at indicated time points and titrated by plaque assay. Data represent the mean ± s.d. of three replicates (Vero E6) and four replicates (hNE). (d) Replication kinetics of recombinant viruses in NhBE at 33°C (left), 37°C (middle) and 39°C (right). NhBE were infected with 100,000 PFU of each virus. Supernatants were collected daily and titrated by TCID50 assay. Data represent the mean ± s.d. of four replicates. ( c-d ) Statistical significance was determined by two-sided unpaired Student’s t -test without adjustments for multiple comparisons. ( c ) P values (left to right): left, NS, P =0.9132; NS P =0.0604; NS P =0.2394; NS P =0.2389; NS P =0.2778; NS P =0.2781; right, NS P =0.1520; NS P =0.3891; NS P =0.9110; NS P =0.8985; NS P =0.1464. ( d ) P values (left to right): left, NS P =0.7943; NS P =0.5025; NS P =0.6683; NS P =0.8985; * P =0.0220; middle, ** P =0.0065; NS P =0.4660; NS P =0.3134; * P =0.0159; right, ** P =0.0094; **** P <10 −4 ; ** P =0.0028; *** P =0.0009. ( e-f ) Competition assay of recombinant viruses in hNE at 33°C and NhBE at 33°C, 37°C and 39°C. The inoculum was prepared by mixing two viruses at 1:1 ratio based on PFU ml −1 and used for infection of hNE and NhBE. Apical wash and supernatant were collected daily, and extracted RNA was used for sequencing. ( e-f ) Bar graph shows proportion of sequencing reads encoding either S-614D or S-614G, and square dots represent individual data points.

Article Snippet: Recombinant hACE2 protein (Cat: RP01266) was purchased from ABclonal.

Techniques: Binding Assay, Fluorescence, Flow Cytometry, Recombinant, Plaque Assay, Infection, Virus, TCID50 Assay, Competitive Binding Assay, Sequencing

Journal: bioRxiv

Article Title: SARS-CoV-2 spike D614G variant confers enhanced replication and transmissibility

doi: 10.1101/2020.10.27.357558

Figure Lengend Snippet: ( a ) Experimental scheme for infection of hACE2-KI mice intranasally infected recombinant SARS-CoV-2 S−614D and SARS-CoV-2 S−614G viruses. Oropharyngeal swabs were sampled daily and tissue samples were analyzed in sub-groups of 4 mice at 2 and 4 days post infection (dpi) in two independent experiments. ( b ) Quantitative RT-PCR analysis of oropharyngeal swabs of inoculated hACE2-KI and wild-type mice. ( c,d ) Pie chart representation of mean frequencies of A or G nucleotide at position 23,403 corresponding to SARS-CoV-2 S−614D and SARS-CoV-2 S−614G , respectively. Each pie chart illustrates the ratio of A/G detected from individual oropharyngeal swab samples (c) and tissues (d) at indicated time post infection. OB, olfactory bulb; ND, not detected.

Article Snippet: Recombinant hACE2 protein (Cat: RP01266) was purchased from ABclonal.

Techniques: Infection, Recombinant, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Antibodies to the SARS-CoV-2 receptor-binding domain that maximize breadth and resistance to viral escape

doi: 10.1101/2021.04.06.438709

Figure Lengend Snippet: a , Phylogenetic relationship of sarbecovirus RBDs inferred from aligned RBD nucleotide sequences, with the four clades of sarbecovirus RBD labeled in separate colors used throughout text. Node support values are rapid bootstrap support values, illustrating substantial ambiguity in the exact relationship between the three clades of sarbecovirus in Asia. b , A yeast-display library containing all sarbecovirus RBDs was assayed for antibody escape analogous to SARS-CoV-2 mutant selections, as shown in . Heatmaps show escape (white) versus binding (black) within the affinity threshold of the FACS escape bins . For S2H97, S2H13, and S2H14, we repeated selections with a more stringent “full escape” bin (0 ng/mL WT control, , ), enabling differentiation of RBDs with intermediate binding (e.g., S2H97/RsSHC014) versus complete loss of binding. c-e , Because the high-throughput assay in ( b ) yields binary measures of binding versus escape at a set threshold determined by FACS gate selection, we performed follow-up quantitative binding assays for select antibody/RBD combinations including flow cytometry detection of antibody binding to isogenic yeast-displayed RBD variants , flow cytometry detection of antibody binding to mammalian-surface displayed spikes ( d ), and ELISA using purified RBD proteins ( e ). These experiments validate the affinity thresholds of “escape” illustrated in ( b ) while adding additional context to interactions that are still present but with reduced binding strength. f , Binding of cross-reactive antibodies (Fab) and human ACE2 to select sarbecovirus RBDs was determined via SPR. NB, no binding; NT, not tested. g , S2H97 neutralization of VSV pseudotyped with select sarbecovirus spikes, with entry measured in VeroE6 (SARS-CoV-2, GD-Pangolin-CoV, and SARS-CoV-1) or ACE2-transduced BHK-21 cells (GX-Pangolin-CoV and WIV1). Curves are representative of at least two independent experiments. Error bars represent standard deviation from three technical replicates from one representative experiment.

Article Snippet: Recombinant hACE2 for SPR (residues 19–615 from Uniprot Q9BYF1 with a C-terminal AviTag-10xHis-GGG-tag, and N-terminal signal peptide) was expressed in HEK293.sus using standard methods (ATUM Bio).

Techniques: Labeling, Mutagenesis, Binding Assay, Control, High Throughput Screening Assay, Selection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Purification, Neutralization, Standard Deviation

Journal: Journal of Agricultural and Food Chemistry

Article Title: Two Resveratrol Oligomers Inhibit Cathepsin L Activity to Suppress SARS-CoV-2 Entry

doi: 10.1021/acs.jafc.2c07811

Figure Lengend Snippet: Screening of entry inhibitors for SARS-CoV-2 using S-pseudovirus. (A) pNL4-3.Luc.R-E- with SARS-CoV-2 S or VSV-G were transfected into 293T cells for 48 h. Then, virions collected from the culture media were analyzed via western blotting using an anti-spike antibody. (B) Expression of hACE2 and β-actin (loading control) in 293T and hACE2/293T cells was measured via western blotting. (C) 293T and hACE2/293T cells were inoculated with S-pseudovirus or S-pseudovirus-infected hACE2/293T cells were treated with neutralizing antibodies of RBD or RBD protein, respectively. 293T cells infected with S-pseudovirus were used as the control. Luciferase activity in cell lysates was measured, and relative fluorescence intensity was calculated. (D,E) hACE2/293T cells were treated with candidate compounds and S-pseudovirus in 96-well plates for 48 h. DMSO-treated cells inoculated with S-pseudovirus were used as the background control. Luciferase activity in cell lysates was measured, and relative fluorescence intensity was calculated. (F) hACE2/293T or 293T cells were treated with candidate compounds and S- or G-pseudovirus for 48 h. DMSO-treated cells inoculated with pseudovirus were used as the control. Luciferase activity in cell lysates was measured, and relative fluorescence intensity was calculated. Experiments were performed in triplicate independently. Data are presented as mean ± SEM of three biological replicates.

Article Snippet: Recombinant hACE2 protein (hACE2-Fc) (Beyotime, Shanghai, China) and RBD (S-RBD-His, Beyotime, Shanghai, China) were incubated with the test compounds or controls at 4 °C for 2 h. Anti-His Magnetic Beads (Beyotime, Shanghai, China) were added to the mixture and incubated for 1 h; the proteins were detected via western blotting.

Techniques: Transfection, Western Blot, Expressing, Control, Infection, Luciferase, Activity Assay, Fluorescence

Journal: Journal of Agricultural and Food Chemistry

Article Title: Two Resveratrol Oligomers Inhibit Cathepsin L Activity to Suppress SARS-CoV-2 Entry

doi: 10.1021/acs.jafc.2c07811

Figure Lengend Snippet: Dose–response experiments of the two resveratrol oligomers on the inhibition of S-pseudovirus entry. (A) Chemical names and structures of the selected entry inhibitors. (B) hACE2/293T cells were treated with various doses of compounds and S-pseudovirus for 48 h. Inhibition rate (●) was quantified by measuring luciferase activity. Cell viability (■) was measured using CCK-8 assay. The CC 50 and IC 50 values were calculated using GraphPad and the curve-fitting method. Experiments were performed in triplicate independently. Data are presented as mean ± SEM of three biological replicates.

Article Snippet: Recombinant hACE2 protein (hACE2-Fc) (Beyotime, Shanghai, China) and RBD (S-RBD-His, Beyotime, Shanghai, China) were incubated with the test compounds or controls at 4 °C for 2 h. Anti-His Magnetic Beads (Beyotime, Shanghai, China) were added to the mixture and incubated for 1 h; the proteins were detected via western blotting.

Techniques: Inhibition, Luciferase, Activity Assay, CCK-8 Assay

Journal: Journal of Agricultural and Food Chemistry

Article Title: Two Resveratrol Oligomers Inhibit Cathepsin L Activity to Suppress SARS-CoV-2 Entry

doi: 10.1021/acs.jafc.2c07811

Figure Lengend Snippet: Effects of the two compounds on the interaction between RBD and hACE2. (A) Purified hACE2-Fc and S-RBD-His proteins were incubated with or without the oligomers for 2 h, and then hACE2-Fc and S-RBD-His were collected via Co-IP and analyzed using western blot. The hACE2-Fc pre-treated anti-ACE2 antibody was used as a positive control. The relative protein levels of hACE2-Fc bound to S-RBD-His were estimated via densitometry. (B) ELISA was used to quantify the inhibition of the interaction between RBD and ACE2. (C) hACE2/293T cells were treated with the compounds for 48 h, and protein expression of hACE2 and β-actin (loading control) were analyzed via western blot. The relative expression of hACE2 to β-actin was estimated via densitometry. Experiments were performed in triplicate independently. Data were presented as mean ± SEM of three biological replicates, and statistical significance was calculated by one-way ANOVA, ns: not significant.

Article Snippet: Recombinant hACE2 protein (hACE2-Fc) (Beyotime, Shanghai, China) and RBD (S-RBD-His, Beyotime, Shanghai, China) were incubated with the test compounds or controls at 4 °C for 2 h. Anti-His Magnetic Beads (Beyotime, Shanghai, China) were added to the mixture and incubated for 1 h; the proteins were detected via western blotting.

Techniques: Purification, Incubation, Co-Immunoprecipitation Assay, Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay, Inhibition, Expressing, Control

Journal: Journal of Agricultural and Food Chemistry

Article Title: Two Resveratrol Oligomers Inhibit Cathepsin L Activity to Suppress SARS-CoV-2 Entry

doi: 10.1021/acs.jafc.2c07811

Figure Lengend Snippet: Effects of compounds on Cat L-mediated entry. (A) Vero E6 or Calu-3 cells were inoculated with S-pseudovirus in the presence of compounds as indicated for 48 h. DMSO-treated cells exposed to pseudovirus were used as a control. Luciferase activity in cell lysates was measured, and relative fluorescence intensity was calculated. (B) Cat L protein was incubated with a fluorogenic substrate (Z-Phe-Arg-7-amido-4-methylcoumarin) in the presence or absence of various concentrations of the test compounds for 1 h in vitro. Luciferase activity of substrate was measured, and relative fluorescence intensity was calculated. (C) hACE2/293T cells were treated with the compounds or Z-FY( t -Bu)-DMK for various periods after S-pseudovirus infection. DMSO-treated cells exposed to pseudovirus were used as the control. Luciferase activity in cell lysates was measured, and relative fluorescence intensity was calculated. Experiments were performed in triplicate independently. Data are presented as mean ± SEM of three biological replicates.

Article Snippet: Recombinant hACE2 protein (hACE2-Fc) (Beyotime, Shanghai, China) and RBD (S-RBD-His, Beyotime, Shanghai, China) were incubated with the test compounds or controls at 4 °C for 2 h. Anti-His Magnetic Beads (Beyotime, Shanghai, China) were added to the mixture and incubated for 1 h; the proteins were detected via western blotting.

Techniques: Control, Luciferase, Activity Assay, Fluorescence, Incubation, In Vitro, Infection

Journal: Journal of Agricultural and Food Chemistry

Article Title: Two Resveratrol Oligomers Inhibit Cathepsin L Activity to Suppress SARS-CoV-2 Entry

doi: 10.1021/acs.jafc.2c07811

Figure Lengend Snippet: Concentration Parameters for Two Compounds in S-pseudovirus Infection Assay

Article Snippet: Recombinant hACE2 protein (hACE2-Fc) (Beyotime, Shanghai, China) and RBD (S-RBD-His, Beyotime, Shanghai, China) were incubated with the test compounds or controls at 4 °C for 2 h. Anti-His Magnetic Beads (Beyotime, Shanghai, China) were added to the mixture and incubated for 1 h; the proteins were detected via western blotting.

Techniques: Concentration Assay, Infection

Journal: Biophysics and Physicobiology

Article Title: E. coli production of a multi-disulfide bonded SARS-CoV-2 Omicron BA.5 RBD exhibiting native-like biochemical and biophysical properties

doi: 10.2142/biophysico.bppb-v20.0036

Figure Lengend Snippet: Binding interaction with the host cell receptor. (a) Binding of the SARS-CoV-2 Omicron BA.5 RBD to the hACE2 using an Octet-N1 Bio-Layer Interferometry. RBD was immobilized on a Ni-NTA sensor chip, and hACE2 was a mobile phase. Control experiments with the reduced (unfolded) RBD on the sensor and hACE2 in the mobile phase (b). Color codes are given within the panel.

Article Snippet: The RBD binding affinity with hACE2 (purity >90% SDS-PAGE) (Bioworld Tech, St Louis Park, Minnesota, USA) was measured using an Octet-N1 Bio-Layer Interferometry (BLI) (Sartorius, Goettingen, Germany).

Techniques: Binding Assay

Journal: Frontiers in Microbiology

Article Title: Mutation-driven parallel evolution in emergence of ACE2-utilizing sarbecoviruses

doi: 10.3389/fmicb.2023.1118025

Figure Lengend Snippet: The SARS-CoV-2 RBD binds to hACE2 via residues located on the three loops. (A) The structure of SARS-CoV-2 RBD and hACE2 complex. The RBM comprising the three loops (designated as RBML1 to RBML3) docks onto the surface of hACE2 (shown in purple; pdb entry 6LZG). (B) LigPlot+ plot of the interaction diagram. Hydrophobic contacts and hydrogen bonding between the two loops (RBML1 and RBML2) of the RBD and the two α-helices (α1 and α2) of hACE2 are shown at the top and the interactions between RBML3 and α1, α13, and the β-hairpin of hACE2 at the bottom. The horizontal dotted line represents the interface, in which the residues involved in direct intermolecular hydrophobic contacts are shown as semicircles with radiating spoke and linked by red dotted lines and hydrogens (<4 Å) are represented by green dashed lines.

Article Snippet: Recombinant hACE2 (Gln18-Ser740) was purchased from KMD Bioscience (Tianjin, China) which was expressed in HEK293 cells with >95% purity.

Techniques:

Journal: Frontiers in Microbiology

Article Title: Mutation-driven parallel evolution in emergence of ACE2-utilizing sarbecoviruses

doi: 10.3389/fmicb.2023.1118025

Figure Lengend Snippet: Structural and dynamics evidences for ACE2 binding origin. (A) Backbone-RMSDs of SARS-CoV-2 RBD and its deletion mutant shown as a function of time (left). Gyrate of proteins. SARS-CoV-2 RBD and its deletion mutant shown as a function of time (right). (B) ΔCα-RMSF data. SARS-CoV-2 RBDCtoS – SARS-CoV-2 RBD is marked in red and SARS-CoV-2 RBDC21_L3 – SARS-CoV-2 RBD in green (left). Conformational ensembles of RBML2 generated by MD simulations and shown by a “sausage” model with MOLMOL (right). (C) A 20-ns MD simulations showing structural dynamics of RBML2 in the apo state or ACE2-bound sate (left). Snapshots extracted from the MD trajectories at 10 and 15 ns, respectively, showing two distinct conformations in RBML2 (open and closed; middle). Comparison of the RBML2 RMSDs between SARS-CoV-2 RBD and the P491T mutant (right). (D) Structural mapping showing parallel molecular evolution removing steric hindrance and electric charge repulse present in the ancestral state. The clash occurs between Pro-502 of RBDs incapable of binding ACE2 and Lys-353 of ACE2, indicated by a cyan dashed circle, and the electric charge repulse between Asp-496 of the RBDs incapable of binding ACE2 and Asp-496 of ACE2, indicated by an orange dashed circle. In the RBD-hACE2 complex, hydrogen bonds are shown by yellow dashed lines and involved residues displayed as sticks.

Article Snippet: Recombinant hACE2 (Gln18-Ser740) was purchased from KMD Bioscience (Tianjin, China) which was expressed in HEK293 cells with >95% purity.

Techniques: Binding Assay, Mutagenesis, Generated, Comparison

Journal: Frontiers in Microbiology

Article Title: Mutation-driven parallel evolution in emergence of ACE2-utilizing sarbecoviruses

doi: 10.3389/fmicb.2023.1118025

Figure Lengend Snippet: Purification, identification and functional characterization of recombinant RBDs. (A) Purification of refolded BtRBD and BtRBD|GY by SEC. Inset: SDS-PAGE analysis of the purified products, marked by a red arrow. “t 6 to t 9 ” denote collection tubes in SEC and “M” denotes protein molecular weight standard. (B) HPLC-Q-TOF-MS determining the molecular mass of BtRBD and BtRBD|GY. (C) Sensorgrams of SARS-CoV-2 RBD binding to the ACE2-immobilized chip surface (left top). The 125 nM analyte concentration was analyzed in duplicate. The concentrations used were 1,000–15.625 nM with two-fold serial dilutions. Sensorgrams of BtRBD to the chip surface (left bottom). The concentrations used were 10,000–625 nM with two-fold serial dilutions. Sensorgrams of BtRBD|GY to the chip surface (right bottom). The concentrations used were 40,000, 10,000, and 2,500 nM. Inset, schematic diagram of SPR experiment, in which the ligand hACE2 was covalently immobilized onto CM5 via its amine groups and the analytes (RBDs) flowed over the surface.

Article Snippet: Recombinant hACE2 (Gln18-Ser740) was purchased from KMD Bioscience (Tianjin, China) which was expressed in HEK293 cells with >95% purity.

Techniques: Purification, Functional Assay, Recombinant, SDS Page, Molecular Weight, Binding Assay, Concentration Assay

Journal: Frontiers in Microbiology

Article Title: Mutation-driven parallel evolution in emergence of ACE2-utilizing sarbecoviruses

doi: 10.3389/fmicb.2023.1118025

Figure Lengend Snippet: The structural basis of the RBML1 of SARS-CoV-2 RBD interacting with hACE2. (A) Gly-446 and Tyr-449 of the RBML1 (colored in cyan) interact with Gln-42 and Asp-38 of hACE2 via three hydrogen bonds (pdb entry 6LZG). In the model of BtRBD, its RBML1 far away from the interface is colored in purple. (B) The lifetimes of the hydrogen bonds during 100-ns MD simulations. The dashed line represents the length threshold (4 Å) of a hydrogen bond.

Article Snippet: Recombinant hACE2 (Gln18-Ser740) was purchased from KMD Bioscience (Tianjin, China) which was expressed in HEK293 cells with >95% purity.

Techniques: